• Humans
• Circulating Tumor DNA
• Workflow
• Cell-Free Nucleic Acids
• Biological Assay
• Neoplasm, Residual

• Biomarkers
• Meta-analysis
• Pancreatic cancer
• Prognosis
• Surgery
• Systematic review

• Breast cancer
• Circulating tumor cells
• Fe(3)O(4)@polydopamine
• MXene@Au@Pt nanozyme
• Multi-aptamer recognition
• Photothermal detection

Liquid biopsies do promise a lot, but are they keeping it? In the past decade, additional novel biomarkers qualified to be called like that, of which, some took necessary hurdles resulting in FDA approval and clinical use. Some others are since a while around, well known and were once regarded to be a game changer in cancer diagnosis or cancer screening. But, during their clinical use limitations were observed from statistical significance and questions raised regarding their robustness, that eventually led to be dropped from associated clinical guidelines for certain applications including cancer diagnosis. The purpose of this review isn't to give a broad overview of all current liquid biopsy as biomarkers, weight them and promise a brighter future in cancer prevention, but rather to take a deeper look on two of those who do qualify to be called liquid biopsies now or then. These two are probably of greatest interest conceptually and methodically, and likely have the highest chances to be in clinical use soon, with a portfolio extension over their original conceptual usage. We aim to dig deeper beyond cancer diagnosis or cancer screening. Actually, we aim to review in depth extracellular vesicles (EVs) and compare with circulating tumour cells (CTCs). The latter methodology is partially FDA approved and in clinical use. We will lay out similarities as taking advantage of surface antigens on EVs and CTCs in case of characterization and quantification. But drawing readers' attention to downstream application based on capture/isolation methodology and simply on their overall nature, here apparently being living material eventually recoverable as CTCs are vs. dead material with transient effects on recipient cell as in case of EVs. All this we try to bring in perspective, compare and conclude towards which future direction we are aiming for, or should aim for. Do we announce a winner between CTCs vs EVs? No, but we provide good reasons to intensify research on them.

• CTC
• biomarker
• ectosomes
• exosomes
• extracellular vesicles
• liquid biopsy
• microvesicles
• personalized medicine

Programmed cell death ligand 1 (PD-L1) inhibitors are commonly used in treating advanced-stage urothelial carcinoma. Contrary to evaluating PD-L1 expression in tumor biopsy samples, this study assessed whether PD-L1 expression in circulating tumor cells (CTCs) can be a predictor of treatment response to PD-L1 inhibitors. The current study proved that there was no statistically significant correlation between the presence of PD-L1-positive CTCs and PD-L1 expression in tumor tissues. Moreover, PD-L1-positive CTCs at baseline could be used as a biomarker to identify patients suitable for PD-L1 blockade therapy. Dynamic changes in PD-L1-positive CTCs during the course of treatment are predictive factors of immunotherapy response and prognostic factors of disease control.

Programmed cell death ligand 1 (PD-L1) inhibitors are commonly used in treating advanced-stage urothelial carcinoma (UC). Therefore, this study evaluated the relationship between PD-L1 expression in circulating tumor cells (CTCs) and treatment response to PD-L1 inhibitors using blood samples collected from patients with UC (n = 23). Subsequently, PD-L1 expression and its clinical correlation were analyzed. All patients had CTCs before PD-L1 inhibitory treatment, of which 15 had PD-L1-positive CTCs. However, PD-L1-positive expression in CTCs was not correlated with PD-L1 expression in tumor biopsy samples. Patients with PD-L1-positive CTCs had better disease control (DC) rates than those without PD-L1-positive CTCs. Moreover, changes in the proportion of PD-L1-positive CTCs were associated with disease outcomes. Furthermore, the PD-L1-positive CTC count in 9 of 11 patients who achieved DC had significantly decreased (p = 0.01). In four patients with progressive disease, this was higher or did not change. PD-L1-positive CTCs at baseline could be used as a biomarker to identify patients suitable for PD-L1 blockade therapy. Dynamic changes in PD-L1-positive CTCs during the course of treatment are predictive factors of immunotherapy response and prognostic factors of disease control. Hence, PD-L1-positive CTCs could be employed as a real-time molecular biomarker for individualized immunotherapy.

• PD-L1
• PD-L1 inhibitors
• circulating tumor cells
• urothelial carcinoma

• Biliary tumors
• Cholangiocarcinoma
• Circulating DNA
• Circulating RNA
• Circulating biomarkers
• Cytokines
• Exosomes
• Liquid biopsy

• ALICE
• cell phenotyping software
• circulating hybrid cells
• hybrid artificial intelligence
• image forgery detection

• disseminated tumor cells
• head and neck squamous cell carcinoma
• micrometastasis
• nanotechnology

• Cancer
• Circulating tumour cells
• Glycosylation
• Lab-on-a-chip
• Liquid biopsy
• Microfluidics

Hepatocellular carcinoma (HCC) is a highly malignant disease with a poor prognosis and high mortality due to a low early diagnosis rate, resistance to systemic treatments and progression to late-stage liver disease. Owing to limitations in the detection of HCC and the lack of awareness of healthcare systems, fewer than 40% of HCC patients are eligible for surgery due to advanced stages of the disease at the time of diagnosis and the occurrence of multiple lesions in the cirrhotic or fibrotic liver. At present, the updated American Association for the Study of Liver Disease (AASLD) guidelines no longer recommend alpha-fetoprotein (AFP) testing as a part of diagnostic evaluation. Thus, it is imperative to establish a novel diagnostic strategy with high sensitivity and reliability to monitor risk factors to detect HCC at an early stage. In recent years, “liquid biopsy,” (including circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA)), has emerged as a technique for the characterization of circulating cells, providing a strong basis for the individualized treatment of patients. As a noninvasive detection method, liquid biopsy is expected to play an important role in the early diagnosis, dynamic monitoring of cancer patients and drug screening. In this review, we will focus on the clinical applications, recent studies and future prospects of liquid biopsy, particularly focusing on HCC.

• Circulating tumor DNA
• Circulating tumor cells
• Hepatocellular carcinoma
• Liquid biopsy

• Bladder cancer
• Circulating tumor cells
• Clinical implications
• Liquid biopsies
• Systematic review
• Technologies

• Biomarkers, Tumor/analysis
• Humans
• Neoplastic Cells, Circulating/pathology
• Precision Medicine
• Urinary Bladder Neoplasms/pathology

• Breast cancer
• CellSearch®
• Circulating tumor cells (CTCs)
• Gastrointestinal tumors
• Lung cancer
• Molecular characterization of single CTCs
• Urological tumors

Single-cell characterization techniques, such as mRNA-seq, have been applied to a diverse range of applications in cancer biology, yielding great insight into mechanisms leading to therapy resistance and tumor clonality. While single-cell techniques can yield a wealth of information, a common bottleneck is the lack of throughput, with many current processing methods being limited to the analysis of small volumes of single cell suspensions with cell densities on the order of 10⁷ per mL. In this work, we present a high-throughput full-length mRNA-seq protocol incorporating a magnetic sifter and magnetic nanoparticle-antibody conjugates for rare cell enrichment, and Smart-seq2 chemistry for sequencing. We evaluate the efficiency and quality of this protocol with a simulated circulating tumor cell system, whereby non-small-cell lung cancer cell lines (NCI-H1650 and NCI-H1975) are spiked into whole blood, before being enriched for single-cell mRNA-seq by EpCAM-functionalized magnetic nanoparticles and the magnetic sifter. We obtain high efficiency (> 90%) capture and release of these simulated rare cells via the magnetic sifter, with reproducible transcriptome data. In addition, while mRNA-seq data is typically only used for gene expression analysis of transcriptomic data, we demonstrate the use of full-length mRNA-seq chemistries like Smart-seq2 to facilitate variant analysis of expressed genes. This enables the use of mRNA-seq data for differentiating cells in a heterogeneous population by both their phenotypic and variant profile. In a simulated heterogeneous mixture of circulating tumor cells in whole blood, we utilize this high-throughput protocol to differentiate these heterogeneous cells by both their phenotype (lung cancer versus white blood cells), and mutational profile (H1650 versus H1975 cells), in a single sequencing run. This high-throughput method can help facilitate single-cell analysis of rare cell populations, such as circulating tumor or endothelial cells, with demonstrably high-quality transcriptomic data.

• Cell Line, Tumor
• Gene Expression Profiling
• High-Throughput Nucleotide Sequencing/methods
• Humans
• Lung Neoplasms/genetics
• Lung Neoplasms/pathology
• Mutation
• RNA, Messenger/genetics

• R33 CA138330 NCI NIH HHS
• U54 CA151459 NCI NIH HHS
• U54 CA199075 NCI NIH HHS
• National Cancer Institute
• National Institutes of Health

• automated cell enumeration.
• breast cancer
• circulating tumor cells
• microfluidic chip

• Algorithms
• Breast Neoplasms/blood
• Cell Line, Tumor
• Female
• HEK293 Cells
• Humans
• Image Processing, Computer-Assisted/methods
• Lab-On-A-Chip Devices
• Microfluidics/instrumentation
• Microfluidics/methods
• Neoplastic Cells, Circulating/pathology

Histology and histopathology are based on the morphometric observations of quiescent cells. Their diagnostic potential could largely benefit from a simultaneous screening of intrinsic biophysical properties at single-cell level. For such a purpose, we analyzed light scattering signatures of individual mononuclear blood cells in microfluidic flow. In particular, we extracted a set of biophysical properties including morphometric (dimension, shape and nucleus-to-cytosol ratio) and optical (optical density) ones to clearly discriminate different cell types and stages. By considering distinctive ranges of biophysical properties along with the obtained relative cell frequencies, we can identify unique cell classes corresponding to specific clinical conditions (p < 0.01). Based on such a straightforward approach, we are able to discriminate T-, B-lymphocytes, monocytes and beyond that first results on different stages of lymphoid and myeloid leukemia cells are presented. This work shows that the simultaneous screening of only three biophysical properties enables a clear distinction between pathological and physiological mononuclear blood stream cells. We believe our approach could represent a useful tool for a label-free analysis of biophysical single-cell signatures.

Paired like homeobox 2B (PHOX2B) is a minimal residual disease (MRD) marker of neuroblastoma. The presence of MRD, also referred to as micro-metastases, is a powerful marker of poor prognosis in neuroblastoma.

Lung metastasis is considered a terminal event in neuroblastoma. Lung micro-metastatic neuroblastoma (MicroNB) cells show high expression levels of PHOX2B and possess a less malignant and metastatic phenotype than lung macro metastatic neuroblastoma (MacroNB) cells, which hardly express PHOX2B.

In vitro assays showed that PHOX2B knockdown in MicroNB cells did not affect cell viability; however it decreased the migratory capacity of the MicroNB-shPHOX2B cells. An orthotopic inoculation of MicroNB-shPHOX2B cells into the adrenal gland of nude mice resulted in significantly larger primary tumors and a heavier micro-metastatic load in the lungs and bone-marrow, than when control cells were inoculated.

PHOX2B expression was found to be regulated by methylation. The PHOX2B promoter in MacroNB cells is significantly more methylated than in MicroNB cells. Demethylation assays using 5-azacytidine demonstrated that methylation can indeed inhibit PHOX2B transcription in MacroNB cells.

These pre-clinical data strongly suggest that PHOX2B functions as a suppressor of neuroblastoma progression.

• PHOX2B
• metastasis
• methylation
• minimal residual disease
• neuroblastoma

• Breast cancer
• circulating tumor cells
• combinational therapy
• drug response
• microfluidics

• Antineoplastic Agents/toxicity
• Biopsy
• Breast Neoplasms/diagnosis
• Breast Neoplasms/metabolism
• Breast Neoplasms/pathology
• Cell Culture Techniques
• Doxorubicin/toxicity
• Female
• Humans
• In Situ Hybridization, Fluorescence
• MCF-7 Cells
• Microfluidic Analytical Techniques
• Neoplasm Metastasis
• Neoplastic Cells, Circulating/drug effects
• Tumor Cells, Cultured

• Mechanobiology Institute
• Singapore-MIT Alliance for Research and Technology

Currently the clinical management of breast cancer relies on relatively few prognostic/predictive clinical markers (estrogen receptor, progesterone receptor, HER2), based on primary tumor biology. Circulating biomarkers, such as circulating tumor DNA (ctDNA) or circulating tumor cells (CTCs) may enhance our treatment options by focusing on the very cells that are the direct precursors of distant metastatic disease, and probably inherently different than the primary tumor's biology. To shift the current clinical paradigm, assessing tumor biology in real time by molecularly profiling CTCs or ctDNA may serve to discover therapeutic targets, detect minimal residual disease and predict response to treatment. This review serves to elucidate the detection, characterization, and clinical application of CTCs and ctDNA with the goal of precision treatment of breast cancer.

• Circulating tumor cells (CTCs)
• biomarker
• cancer
• cell free DNA (cfDNA)
• circulating tumor DNA (ctDNA)

Analysis of circulating tumor cells from patients with different types of cancer is nowadays a fascinating new tool of research and their number is proven to be useful as a prognostic factor in metastatic breast, colon and prostate cancer patients. Studies are going beyond enumeration, exploring the circulating tumor cells to better understand the mechanisms of tumorigenesis, invasion and metastasis and their value for characterization, prognosis and tailoring of treatment. Few studies investigated the prognostic significance of circulating tumor cells in germ cell tumors. In this review, we examine the possible significance of the detection of circulating tumor cells in this setting.

• biomarkers
• circulating tumor cells
• germ cell tumors

• Cancer stem cells
• circulating tumor cells (CTCs)
• drug resistance

Colon cancer development and malignant progression are driven by genetic and epigenetic alterations in tumor cells and by factors from the tumor microenvironment. Cancer cells become reliant on the activity of specific oncogenes and on prosurvival and proliferative signals they receive from the abnormal environment they create and reside in. Accordingly, the response to anticancer therapy is determined by genetic and epigenetic changes that are intrinsic to tumor cells and by the factors present in the tumor microenvironment. Recent advances in the understanding of the involvement of the tumor microenvironment in tumor progression and therapeutic response are optimizing the application of prognostic and predictive factors in colon cancer. Moreover, new targets in the tumor microenvironment that are amenable to therapeutic intervention have been identified. Because stromal cells are with rare exceptions genetically stable, the tumor microenvironment has emerged as a preferred target for therapeutic drugs. In this review, we discuss the role of stromal fibroblasts and macrophages in colon cancer progression and in the response of colon cancer patients to therapy.

• colon cancer
• fibroblasts
• macrophages
• tumor microenvironment

• Animals
• Cell Line, Tumor
• Cell Separation/instrumentation
• Cell Separation/methods
• Cell Survival
• Disease Models, Animal
• Humans
• Mice
• Micropore Filters
• Neoplastic Cells, Circulating/pathology
• Reproducibility of Results

• DP2 CA174508 NCI NIH HHS
• DP2CA174508 NCI NIH HHS

• Biomarkers, Tumor/analysis
• Humans
• Neoplasm Proteins/analysis
• Neoplastic Cells, Circulating

• Adult
• Aged
• Antigens, CD/blood
• Antigens, CD/metabolism
• Antigens, Differentiation, Myelomonocytic/blood
• Antigens, Differentiation, Myelomonocytic/metabolism
• Antigens, Neoplasm/blood
• Antigens, Neoplasm/metabolism
• Biomarkers, Tumor/blood
• Biomarkers, Tumor/metabolism
• Breast Neoplasms/blood
• Breast Neoplasms/metabolism
• Breast Neoplasms/pathology
• Cell Adhesion Molecules/blood
• Cell Adhesion Molecules/metabolism
• Cell Line, Tumor
• Epithelial Cell Adhesion Molecule
• ErbB Receptors/blood
• ErbB Receptors/metabolism
• Female
• Flow Cytometry
• Humans
• Immunohistochemistry
• Keratin-18/blood
• Keratin-18/metabolism
• Keratin-19/blood
• Keratin-19/metabolism
• Keratin-8/blood
• Keratin-8/metabolism
• Leukocyte Common Antigens/blood
• Leukocyte Common Antigens/metabolism
• MCF-7 Cells
• Microscopy, Confocal
• Middle Aged
• Neoplasm Metastasis
• Neoplastic Cells, Circulating/metabolism
• Prognosis
• Prospective Studies
• Vimentin/blood
• Vimentin/metabolism

• UL1 TR001070 NCATS NIH HHS
• UL1 TR000090 NCATS NIH HHS
• UL1RR025755 NCRR NIH HHS
• 5 P30 CA16058-26 NCI NIH HHS
• R01 CA062349 NCI NIH HHS
• UL1 RR025755 NCRR NIH HHS
• R01 CA97391-01A1 NCI NIH HHS

• antigen-independent
• cancer
• circulating tumor cells
• enrichment
• physical properties

• Cell Physiological Phenomena
• Cell Separation/instrumentation
• Cell Separation/methods
• Chemical Phenomena
• Humans
• Neoplastic Cells, Circulating

• DP2 CA174508 NCI NIH HHS
• R21 CA161835 NCI NIH HHS
• DP2CA174508 NCI NIH HHS
• R21CA161835 NCI NIH HHS

• disseminated tumor cells
• dormant tumor cells
• metastasis
• microenvironment
• micrometastasis
• neuroblastoma

• Animals
• Apoptosis/genetics
• Biomarkers, Tumor/metabolism
• Cell Cycle Checkpoints/genetics
• Cell Line, Tumor
• Cell Movement/genetics
• Cell Survival/genetics
• Disease Progression
• Focal Adhesion Kinase 1/genetics
• Focal Adhesion Kinase 1/metabolism
• Humans
• Lung Neoplasms/genetics
• Lung Neoplasms/metabolism
• Lung Neoplasms/pathology
• Lung Neoplasms/secondary
• MAP Kinase Signaling System/genetics
• Male
• Mice
• Mice, Inbred BALB C
• Mice, Nude
• Neoplasm Metastasis
• Neuroblastoma/genetics
• Neuroblastoma/metabolism
• Neuroblastoma/pathology
• Neuroblastoma/secondary
• Phosphorylation/genetics
• Stem Cells/metabolism
• Stem Cells/pathology
• Tumor Microenvironment

• BRMS1
• CTC
• adhesion
• integrins
• metastasis

• Biomarker
• CTC detection methods
• CTC isolation methods
• Cancer
• Carcinoma
• Circulating tumor cell
• Image cytometry
• Immunomagnetic
• Metastasis
• Microfluidics

Circulating tumor cells (CTCs) are prognostic markers in a variety of solid tumor malignancies. The potential of CTCs to be used as a “liquid biopsy” to monitor a patient’s condition and predict drug response and resistance is currently under investigation. Using a negative depletion, enrichment methodology, CTCs isolated from the peripheral blood of breast cancer patients with stage IV breast cancer undergoing DNA damaging therapy with platinum-based therapy were enriched. The enriched cell suspensions were stained with an optimized labeling protocol targeting: nuclei, cytokeratins 8, 18, and 19, the surface marker CD45, and the presence of the protein γ-H2AX. As a direct or indirect result of platinum therapy, double-strand break of DNA initiates phosphorylation of the histone H2AX, at serine 139; this phosphorylated form is referred to as γ-H2AX. In addition to γ-H2AX staining in specific locations with the cell nuclei, consistent with previous reports and referred to as foci, more general staining in the cell cytoplasm was also observed in some cells suggesting the potential of cell apoptosis. Our study underscores the utility and the complexity of investigating CTCs as predictive markers of response to various therapies. Additional studies are ongoing to evaluate the diverse γ-H2AX staining patterns we report here which needs to be further correlated with patient outcomes.

• Her2neu
• chemotherapy
• circulating tumor cells
• metastatic breast cancer
• γ-H2AX

Osteosarcoma (OS) is the most common malignant bone tumor and the prognosis depends on pulmonary metastases, which arise from multi-step progression of malignant tumors. We herein aimed to clarify the critical step of pulmonary metastasis using the syngeneic mouse spontaneous highly metastatic OS LM8 and parental Dunn cell lines, to identify new candidate molecules to suppress pulmonary metastasis. We first investigated the chronological detection of circulating tumor cells (CTCs) from mice with either cell line. LM8 CTCs appeared faster, at a higher rate and with a greater number compared to Dunn CTCs. Cultured cells from CTCs of LM8 showed higher proliferative ability than cells from the primary site in suspension culture, which mimicked the environment of the bloodstream for CTCs. The proliferative ability of LM8 cells was also higher than that of Dunn cells in 3D collagen culture with low stiffness (−150 Pa; close to conditions in the lung). We next focused on the extravasation step. LM8 showed higher migration ability compared to Dunn with transendothelial migration assay. We also found a disruption in endothelial barrier function throughout co-culture with LM8 using time-lapse imaging. In addition, LM8 secreted high levels of vascular endothelial growth factor (VEGF), while VEGF signal inhibition with a small molecule tyrosine kinase inhibitor (pazopanib) decreased disruption of the vascular barrier and transendothelial migration of LM8. Finally, daily oral administration of pazopanib reduced the rate and size of pulmonary metastasis in vivo. Collectively, these results show anti-VEGF therapy as a candidate for pulmonary metastasis of OS.

• Humans
• Immunomagnetic Separation/instrumentation
• Immunomagnetic Separation/methods
• Neoplasms/pathology
• Neoplastic Cells, Circulating

• R01 CA062349 NCI NIH HHS
• UL1 RR025755 NCRR NIH HHS

• Animals
• Bone Morphogenetic Protein 7/metabolism
• Bone Morphogenetic Protein Receptors, Type II/biosynthesis
• Bone Neoplasms/metabolism
• Bone Neoplasms/pathology
• Bone Neoplasms/secondary
• Bone Neoplasms/therapy
• Cell Cycle Proteins/biosynthesis
• Cyclin-Dependent Kinase Inhibitor p27/biosynthesis
• Gene Expression Regulation, Neoplastic
• Humans
• Intracellular Signaling Peptides and Proteins
• Male
• Mice
• Neoplasm Metastasis
• Neoplasm Proteins/metabolism
• Neoplasm Transplantation
• Neoplastic Stem Cells/metabolism
• Neoplastic Stem Cells/pathology
• Prostatic Neoplasms/metabolism
• Prostatic Neoplasms/pathology
• Prostatic Neoplasms/therapy
• Transplantation, Heterologous
• p38 Mitogen-Activated Protein Kinases/metabolism

• R01 CA124650 NCI NIH HHS
• R01 CA129000 NCI NIH HHS
• R01CA129000 NCI NIH HHS
• R01CA124650 NCI NIH HHS

• Animals
• Apoptosis
• Biomarkers, Tumor/metabolism
• Blotting, Western
• Bone Marrow Neoplasms/metabolism
• Bone Marrow Neoplasms/secondary
• Cell Movement
• Cell Proliferation
• Flow Cytometry
• Homeodomain Proteins/metabolism
• Humans
• Lung Neoplasms/metabolism
• Lung Neoplasms/secondary
• Male
• Matrix Metalloproteinases/metabolism
• Mice
• Mice, Inbred BALB C
• Mice, Nude
• Mitogen-Activated Protein Kinase 3/metabolism
• Neoplasm, Residual/metabolism
• Neoplasm, Residual/pathology
• Neuroblastoma/metabolism
• Neuroblastoma/pathology
• Phosphorylation
• Proto-Oncogene Proteins c-akt/metabolism
• RNA, Messenger/genetics
• Reverse Transcriptase Polymerase Chain Reaction
• Transcription Factors/metabolism
• Tumor Cells, Cultured
• Tyrosine 3-Monooxygenase/metabolism

• Biomarkers, Tumor/blood
• Humans
• Melanoma/blood
• Melanoma/diagnosis
• Neoplastic Cells, Circulating
• Skin Neoplasms/blood
• Skin Neoplasms/diagnosis